BHK cells (supplied by Novo Nordisk) transfected with the inducible GluR6 ion channel were grown as monolayers in 75 cm2 flasks at 37 C in a humidified 5% CO2 incubator. GluR6 expression was induced in the cells with 5 mM (final concentration) isopropyl- 1-thio-β-D-galactopyranoside (IPTG). All dilutions of the agonist, antagonist and [14C] guanidinium (from Amersham Pharmacia Biotech) were prepared in assay buffer, consisting of NMG buffer containing 10 mM HEPES, pH 7.5, 2 mM CaCl2, 150 mM Nmethyl- D-glucamine and Ringers buffer containing 10 mM HEPES, pH 7.5, 150 mM NaCl, 3 mM KCl, 1 mM CaCl2 and 20 mM sucrose. 4 parts NMG and 1 part Ringers buffer were prepared containing an additional 2.2 mM CaCl2, to which was added 500 μM procaine and 1 mg/ml concanavalin A, to prevent receptor desensitization.
ADDITION OF THE AGONIST DOMOATE USING THE MICROBETA JET
Assays were set up to compare the effect of adding the agonist manually to the wells of Cytostar-T microplates and adding the agonist through the injectors of the MicroBeta JET. Duplicate wells were assayed containing 50 μl [14C] guanidinium (10 μCi/ml) to which was added 50 μl of the prepared agonist. No agonist control wells were also assayed. These contained assay buffer instead of the agonist. After all additions were completed, replicate wells were counted simultaneously on different detectors for 20 secs/well using repeated counting cycles.
ADDITION OF THE ANTAGONIST KYNURENIC ACID USING THE MICROBETA JET
The effect of the antagonist kynurenic acid was detrmined on the GluR6 assay system. Duplicate wells were assayed containing 50 μl [14C] guanidinium and either 0.1 μM domoate or 0.1 μM domoate/